Peroxisomes: 40 years of histochemical staining,personal reminiscences

The historical circumstances that led to the discovery of the 3,3′-diamino-benzidine (DAB) method for staining of peroxisomes 40 years ago are reviewed. In the course of studies on the uptake and absorption of horse radish peroxidase in mammalian liver, in sections incubated for detection of peroxidase activity in DAB, it was noted that peroxisomes also stained positively for peroxidase activity. Subsequently, it was revealed that the peroxidatic activity of catalase, which is abundantly present in peroxisomes, is responsible for that staining. This notion was confirmed in quantitative biochemical studies with crystalline beef liver catalase and in tracer studies using catalase as an ultrastructural tracer. The application of the DAB method led to the discovery of peroxisomes as a ubiquitous eukaryotic cell organelle, attracting great interest in their investigation in biomedical research.     

Small interfering RNA–mediated gene suppression as a therapeutic intervention in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the lethal and difficult-to-cure cancers worldwide. Owing to the late diagnosis and drug resistance of malignant hepatocytes, treatment of this cancer by conventional chemotherapy agents is challenging, and researchers are seeking new alternative treatment options to overcome therapy resistance in this neoplasm. RNA interference (RNAi) is a potent and specific approach in targeting gene expression and has emerged as a novel therapeutic tool for many diseases, including cancers. Small interfering RNA (siRNA) is a type of RNAi that is produced intracellularly from exogenous synthetic oligonucleotides and can selectively knock down target gene expression in a sequence-specific manner. Various factors play roles in the initiation and progression of HCC and provide multiple candidate targets for siRNA intervention. In addition, due to the liver's unique architecture and availability of some hepatic siRNA delivery methods, this organ has received much more attention as a target tissue for such oligonucleotide action. Recent advances in designing nanoparticle systems for the in vivo delivery of siRNAs have markedly enhanced the potency of siRNA-mediated gene silencing under clinical development for HCC therapy. The utility of siRNAs as anti-HCC agents is the subject of the current review. siRNA-based gene therapies could be one of the main feasible approaches for HCC therapy in the future.     

Three-dimensional ultrastructural analysis of peroxisomes in HepG2 cells

Peroxisomes in the human hepatoblastoma cell line, HepG2, exhibit distinct alterations of shape, size, and distribution, dependent on culture conditions (cell density, duration in culture, and presence of specific growth factors). Although many cells with elongated tubular peroxisomes are present in thinly seeded cultures, spherical particles forming large focal clusters are found in confluent cultures. The authors have analyzed the ultrastructure and the spatial relationship of peroxisomes of HepG2 cells at different stages of differentiation, using three-dimensional (3D)-reconstruction of ultrathin serial sections, and electronic image processing. Cells were prepared for immunofluorescence using different antibodies against peroxisomal matrix and membrane proteins, as well as for electron microscopy after the alkaline 3,3′-diaminobenzidine staining for catalase. The results indicate that the tubular peroxisomes, which can reach a length of several microns, are consistently isolated, and never form an interconnected peroxisomal reticulum. At the time of disappearance of tubular peroxisomes, rows of spherical peroxisomes, arranged like beads on a string, are observed, suggesting fission of tubular ones. In differentiated confluent cultures, clusters of several peroxisomes are seen, which, by immunofluorescence, appear as large aggregates, but after 3D reconstruction consist of single spherical and angular peroxisomes without interconnections. The majority of such mature spherical peroxisomes (but not the tubular ones) exhibit tail-like, small tubular and vesicular attachments to their surface, suggesting a close functional interaction with neighboring organelles, particularly the endoplasmic reticulum, which is often observed in close vicinity of such peroxisomes.     

The Inhibition Effect of Lactobacilli Against Growth and Biofilm Formation of <Emphasis Type="BoldItalic">Pseudomonas aeruginosa</Emphasis>

The emergence of antibiotic-resistant and food-spoilage microorganisms has renewed efforts to identify safe and natural alternative agents of antibiotics such as probiotics. The aim of this study was the isolation of lactobacilli as potential probiotics from local dairy products with broad antibacterial and anti-biofilm activities against antibiotic-resistant strains of Pseudomonas aeruginosa and determination of their inhibition mechanism. Antibiotic susceptibility and classification of acquired resistance profiles of 80 P. aeruginosa strains were determined based on Centers for Disease Control and Prevention (CDC) new definition as multidrug-resistant (MDR), extensively drug-resistant (XDR), and pan-drug-resistant (PDR) followed by antibacterial assessment of lactobacilli against them by different methods. Among the 80 P. aeruginosa strains, 1 (1.3%), 50 (62.5%), and 78 (97.5%) were PDR, XDR, and MDR, respectively, and effective antibiotics against them were fosfomycin and polymyxins. Among 57 isolated lactobacillus strains, two strains which were identified as Lactobacillus fermentum using biochemical and 16S rDNA methods showed broad inhibition/killing and anti-biofilm effects against all P. aeruginosa strains. They formed strong biofilms and had bile salts and low pH tolerance. Although investigation of inhibition mechanism of these strains showed no bacteriocin production, results obtained by high-performance liquid chromatography (HPLC) analysis indicated that their inhibitory effect was the result of production of three main organic acids including lactic acid, acetic acid, and formic acid. Considering the broad activity of these two L. fermentum strains, they can potentially be used in bio-control of drug-resistant strains of P. aeruginosa.     

Cardiolipin Interactions with Proteins

Cardiolipins (CL) represent unique phospholipids of bacteria and eukaryotic mitochondria with four acyl chains and two phosphate groups that have been implicated in numerous functions from energy metabolism to apoptosis. Many proteins are known to interact with CL, and several cocrystal structures of protein-CL complexes exist. In this work, we describe the collection of the first systematic and, to the best of our knowledge, the comprehensive gold standard data set of all known CL-binding proteins. There are 62 proteins in this data set, 21 of which have nonredundant crystal structures with bound CL molecules available. Using binding patch analysis of amino acid frequencies, secondary structures and loop supersecondary structures considering phosphate and acyl chain binding regions together and separately, we gained a detailed understanding of the general structural and dynamic features involved in CL binding to proteins. Exhaustive docking of CL to all known structures of proteins experimentally shown to interact with CL demonstrated the validity of the docking approach, and provides a rich source of information for experimentalists who may wish to validate predictions.     

Modeling the time dependent distribution of a new 153Sm complex for targeted radiotherapy purpose

BackgroundFor radioimmunotherapy purposes, a chemical complex with high absorption in cancer tumor is required. New chemicals are to be examined for their concentration in tumor and healthy organs. These are labeled with β-emitting radioisotopes to irradiate the tumor while deposited inside it.AimTo study the capability of recently developed chemical complex in targeting cancer tumor and investigate the distribution of ¹⁵³Sm-TPTTC in rat organs as function of time.Materials and methodsThe chemical complex – [Tris(1,10-phenanthroline)Samarium(III)] trithiocyanate was prepared and labeled with ¹⁵³Sm radioisotope. The labeled complex was injected to a population of tumor bearing mice. In 2, 4, 24, 48, 96 h after injection the animals were sacrificed and the concentration of Samarium complex was measured in various organs such as blood, heart, intestine, colon, liver, spleen, kidney, sternum and bone.ResultsThe concentration of the radiopharmaceutical in various organs was measured at different times. The temporal behavior of biodistribution of ¹⁵³Sm-TPTTC was modeled and drawn as function of time.ConclusionIt is shown that ¹⁵³Sm-TPTTC is concentrated in tumor tissue and liver much more than in other organs. The variation of pharmaceutical concentration in all organs is described with summation of eight exponential terms and it approximates our experimental data with precision better than 2%.     

Activators of the energy sensing kinase AMPK inhibit random cell movement and chemotaxis in U937 cells

AMP-activated protein kinase (AMPK) is a key energy sensor, known to regulate energy metabolism in diverse cell types. Hypoxia is encountered frequently in the microenvironments of inflammatory lesions and is a critical regulator of function in inflammatory cells. Energy deficiency is one of the consequences of hypoxia, but its potential role in modulating leucocyte function has received little attention. Using micropore chemotaxis assays to assess migratory responses of the monocyte-like cell line U937, it was found that the AMPK activators AICAR and phenformin rapidly reduced random migration (chemokinesis) as well as chemotaxis due to stromal cell-derived factor (SDF)1alpha. There was an approximate 50% reduction in both chemokinesis and chemotaxis following 30 min preincubation with both AICAR and phenformin (P < 0.01), and this continued with up to 24 h preincubation. The binding of SDF1alpha to its receptor CXCR4 was unaltered, suggesting AMPK was acting on downstream intracellular signalling pathways important in cell migration. As AMPK and statins are known to inhibit HMG CoA reductase, and both reduce cell migration, the effect of mevastatin on U937 cells was compared with AMPK activators. Mevastatin inhibited cell migration but required 24 h preincubation. As expected, the inhibitory effect of mevastatin was associated with altered subcellular localization of the Rho GTPases, RhoA and cdc42, indicating decreased prenylation of these molecules. Although the effect of AMPK activation was partially reversed by mevalonate, this was not associated with altered subcellular localization of Rho GTPases. The data suggest that activation of AMPK has a general effect on cell movement in U937 cells, and this is not due to inhibition of HMG CoA reductase. These are the first data to show an effect of AMPK on cell movement, and suggest a fundamental role for energy deficiency in regulating cellular behaviour.     

Indole-3-Acetic Acid (IAA) Production in Symbiotic and Non-Symbiotic Nitrogen-Fixing Bacteria and its Optimization by Taguchi Design

Production of Indole-3-acetic acid (IAA) in 35 different symbiotic and non-symbiotic nitrogen-fixing bacteria strains isolated from soil and plant roots was studied and assayed by chromatography and colorimetric methods. These bacteria included Agrobacterium, Paenibacillus, Rhizobium, Klebsiella oxytoca, and Azotobacter. The best general medium and synergism effects of isolates for IAA production were investigated. Effects of different variables containing physical parameters and key media components and optimization of condition for IAA production were performed using the Design of Experiments. Qualitek-4 (W32b) software for automatic design and analysis of the experiments, both based on Taguchi method was used. The results showed that Rhizobium strains, symbiotic, and Paenibacillus non-symbiotic bacteria yielded the highest concentrations of IAA (in the range of 5.23–0.27 and 4.90–0.19 ppm IAA/mg biomass, respectively) and IAA production was increased by synergism effect of them. Yeast Extract Mannitol medium supplemented with l-tryptophan was the best general medium for IAA production. The analysis of experimental data using Taguchi method indicated that nitrogen source is very prominent variable in affecting the yield and mannitol as carbon source, potassium nitrate (1%), and l-tryptophan (3 g/l) as nitrogen sources after 72-h incubation at 30°C were the optimum conditions for production of IAA. 5.89 ppm IAA/mg biomass was produced under these optimal conditions.